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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells
  • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/57—IFN-gamma

Definitions

• TRF T lymphocyte replacing factor
• the thus obtained lymphokine producing T lymphocyte hybrid may be cultured in a scale of 10 - 20 ml in a conventional tissue culture medium using a Petri dish or a flask.
• a shaking culture, rotatory culture, stirred culture etc. may be carried out with or without aeration by air containing C0 2 gas.
• a mass-culture is not possible with an ordinary static culture.
• T lymphocyte-derived hybridoma which produces a lymphokine may be cultured in a large scale by employing any of (a), (b) and (c) or a combination thereof and other conditions . which generally permit the supply of adequate oxygen to the cells by enhancing the amount of oxygen dissolved in the medium.
• the T lymphocyte hybridoma requires a stimulant such as phytohemmagglutinin, concanavalin A, pokeweed mitogen, protein A, bacterial cells and components derived therefrom etc. for the production of the lymphokine
• the stimulant may be added to the medium when the proliferation of the cells has reached the predetermined cell concentration.
• the production of the lymphokine from the hybridoma may be enhanced by adding a phorbol ester, hydroxyurea, interleukin 1 etc., as required, at an appropriate concentration to the medium.
• Table 3 shows the cell proliferation in the culture modes E and F.
• the roller bottle was employed for steadily supplying fresh oxygen to the medium and rotatory culture was conducted as in F, the hybridoma exhibited favorable proliferation.
• E where culture was conducted without rotating the roller bottle, the cells did not proliferate at all.
• a commercially available ICR mouse was intraperitoneally administered with one ml of physiological saline containing 10% by weight of proteose peptone, and 4 days later, the peritoneal exudate cells were collected in conventional manner well known in the art.
• the cells were adjusted to 8 x 10 5 cells/ml and allotted 1 ml each to the respective wells on a 24-welled tissue culture plate, followed by culture for 3 'hours.
• the unattched cells were washed away with the medium to leave only the attached cells, and 1 ml of a sample to be measured for the M A F activity was added.
• the sample was removed, the well was washed once with the medium, and 100 ⁇ 1 of a suspension of P815 cells (cells generally employed in the technical field of the invention) adjusted to 2 x 10 5 cells/ml was added. After 20 hours of culture, 1 ⁇ Ci of tritium-labeled thymidine was added and culture was continued for further 4 hours, after which the cells were harvested in a conventional manner well known in the art, and the radtio- activity uptake by the P815 cells was measured. Since the attached cells activated by MAF (phagocytes) inhibit the DNA synthesis by P815, the presence of the MAF activity in the hybridoma culture supernatant may be easily judged.
• MAF phagocytes
• the obtained hybridomas were each cultured according to the method in (b) of Example 1, and each culture supernatant was collected. This culture supernatant was tested for the IFN (hereinafter sometimes referred to as IFN) activity according to the following method, to judge whether ⁇ -interferon (hereinafter sometimes referred to as ⁇ -IFN) had been spontaneously produced.
• IFN IFN
• ⁇ -IFN ⁇ -interferon
• F13-K15-I126 was cultured according to the methods C and F in (d) of Example 1, then on the third day and fourth day, each culture super-natant was collected and the produced ⁇ -IFN activity was measured according to the ⁇ -IFN activity testing method described in (c).

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Cited By (6)

• 1982
  • 1982-10-19 EP EP82109658A patent/EP0077571A3/de not_active Withdrawn

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